Abstract

Background and Aims

The gametophytes of most homosporous ferns are cordate–thalloid in shape. Some are strap- or ribbon-shaped and have been assumed to have evolved from terrestrial cordate shapes as an adaptation to epiphytic habitats. The aim of the present study was to clarify the morphological evolution of the strap-shaped gametophyte of microsoroids (Polypodiaceae) by precise analysis of their development.

Methods

Spores of Colysis decurrens collected in Kagoshima, Japan, were cultured and observed microscopically. Epi-illuminated micrographs of growing gametophytes were captured every 24 h, allowing analysis of the cell lineage of meristems. Light microscopy of resin-sections and scanning electron microscopy were also used.

Key Results

Contrary to previous assumptions that strap-shaped Colysis gametophytes have no organized meristem, three different types of meristems are formed during development: (1) apical-cell based – responsible for early growth; (2) marginal – further growth, including gametophyte branching; and (3) multicellular – formation of cushions with archegonia. The cushion is two or three layers thick and intermittent. The apical-cell and multicellular meristems are similar to those of cordate gametophytes of other ferns, but the marginal meristem is unique to the strap-shaped gametophyte of this fern.

Conclusions

The strap-shaped gametophytes of C. decurrens may have evolved from ancestors with a cordate shape by insertion of the marginal meristem phase between the first apical-cell-based meristem and subsequent multicellular meristem phases. Repeated retrieval of the marginal meristem at the multicellular meristem phase would result in indefinite prolongation of gametophyte growth, an ecological adaptation to epiphytic habitats.

INTRODUCTION

Pteridophytes are characterized by having two independent plant bodies, sporophytes and gametophytes. It is plausible that both sporophytes and gametophytes have been subjected to similar environmental conditions and then adapted to the habitats where they grow (Watkins et al., 2007). As discussed by Dassler and Farrar (1997), data on gametophytes are critical in understanding pteridophyte reproduction, distribution, ecology and evolution, but nevertheless adaptive morphology in gametophytes has been largely ignored by researchers until recently.

Although gametophytes of homosporous ferns are generally cordate-thalloid with a midrib (cushion), some gametophytes are strap- or ribbon-shaped or filamentous (Bower, 1923; Orth, 1936; Nayar and Kaur, 1971). Strap- and ribbon-shaped gametophytes are found in members of the Hymenophyllaceae, vittarioids and Polypodiaceae, suggesting parallel evolution in different clades of ferns as an adaptation to epiphytic habitats (Farrar et al., 2008). Therefore, the study of the evolution of gametophytes adapted to epiphytic habitats may provide useful data for a better understanding of the adaptive evolution of fern sporophytes.

Nayar and Kaur (1969) claimed that strap- and ribbon-shaped gametophytes have no organized meristems, unlike ordinary cordiform gametophytes, but their conclusion was based on a number of gametophytes fixed at different developmental stages, not from long-term observations of the same gametophyte. Cell lineage analyses focusing on meristem behaviour in growing gametophytes are needed to clarify the development and evolution of gametophytes. In the present study, a new sequential observation technique with epi-illuminated micrographs of gametophytes growing in culture was used, permitting precise cell lineage analysis.

Microsoroids, which are one of four clades of Polypodiaceae (Schneider et al., 2004), produce various shaped gametophytes depending on genera or species: strap-shaped (Colysis, Nayar, 1962), ribbon-shaped (Leptochilus and Paraleptochilus, Nayar 1963b), and cordiform or elongate cordiform gametophytes (Microsorium, Nayar, 1963a; Microsorum (Microsorium), Pal and Pal, 1962). Microsoroids grow in epilithic (rock surfaces), epiphytic and also terrestrial habitats (Hennipman et al., 2000). Thus, microsoroids have a key role to play in clarifying the evolution of adaptive morphologies of gametophytes in relation to habitat preferences. The aim of the present study was to reveal the evolutionary course of strap-shaped gametophytes in microsoroids. Colysis decurrens grows in both epilithic and terrestrial habitats, and has strap-shaped gametophytes (Momose, 1967). The development of C. decurrens gametophytes was examined in comparison with cordiform gametophytes of other ferns.

The morphology of growing gametophytes was recorded by sequential observation in which microscopic images of growing gametophytes were captured using a metallurgical microscope (Nikon-OPTIPHOT-2, Nikon Co., Tokyo, Japan) with epi-illumination roughly every 24 h. Images at several focal points of a gametophyte were synthesized into a clear image using All-in-Focus DM-99i II (Synoptics Ltd, Cambridge, UK) and Photoshop CS (Adobe Systems Inc., San Jose, CA, USA). Comparison of one image with the preceding image allowed tracing of cell lineages. Thirty-seven individuals at early developmental stages, i.e. vegetative stage, and around 20 individuals at later developmental stages were observed in detail. There was no difference in the development of gametophytes grown from spores collected from the two localities mentioned above.

For anatomical observations, materials fixed with FAA were dehydrated through a graded ethanol series, embedded in Historesin (glycol methacrylate, Leica, Heidelberg, Germany), cut into 2-μm-thick sections, and stained with modified Sharman's solution (Jernstedt et al., 1992). To compare cell size in or between meristems of gametophytes at different developmental stages, the length and thickness of six cells (outermost and five inner cells behind) were measured in three serial longitudinal sections (100 µm apart) of a gametophyte. Five and ten individuals, respectively, were used for measurement of cell size at the marginal and cushion meristem phases. Another individual with a newly initiating cushion meristem was used for cell size measurement.

RESULTS

Early development of gametophytes with apical-cell-based meristems

About 10 d after spore sowing (DAS), a filament about four cells long forms as a result of cell division by primary walls (wall I), which are perpendicular to the long axis of the filament (Fig. 1A, B). The terminal and sub-terminal cells of these four cells of the filamentous gametophyte divide similarly by the secondary wall (wall II) at right angles to wall I, resulting in an anterior half composed of four cells, each of which is a quadrant (data not shown). In contrast, the posterior half of the filament does not divide further, but forms rhizoids (Fig. 1C, D). The terminal two quadrants, which are side-by-side, each undergo periclinal (horizontal) and anticlinal (vertical) divisions by wall III and IV, producing triangular cells in both lateral sides of the anterior half (Fig. 1C–F).

The right and left halves separated by wall II in the anterior semicircular plate (Fig. 1E, F) soon become different in shape and overall size. In Fig. 1E and F, the half demarcated by walls I and II has slightly more cells than the other half, involving the triangular cell with its own derivative cell. The triangular cell (a) in one half continues to function as an apical cell, dividing alternately at two lateral walls to produce derivative cells that undergo periclinal and anticlinal divisions to form rectangular cell packets (merophytes) (Fig. 1G–J). In contrast, the other triangular cell in the other half soon ceases division and its surrounding cells divide much less frequently (Fig. 1G–J). After this stage, gametophyte growth is mainly from the single apical cell and to merophytes derived from it, and the gametophyte becomes ovobate in form (Fig. 1K, L).

Either one of the two triangular cells can become the apical cell. However, sometimes (six of 37 individuals investigated here) neither triangular cell differentiates into the apical cell, producing a few derivative cells (Fig. 1O–R). Instead, one of the rectangular surface cells between the triangular cells becomes the apical cell through two continuous oblique divisions (Fig. 1Q–T). In this case, cells demarcated by walls II and IV contribute to most of the gametophyte.

Growing gametophytes are inclined at around 45° from the original long axis of the filamentous gametophyte due to activity of the apical cell formed at the lateral side of the anterior part of the young gametophyte (Fig. 1I, J). This type of development occurred in 15 of 37 individuals but not in 16 others where wall I in the anterior half is oblique and the just-formed apical cell is at the apex of the growing gametophyte rather than the lateral side (Fig. 1U, V). The triangular cell, which fails to become the apical cell, produces no derivative cells (Fig. 1X). In this case, the gametophyte grows vertically along the original long axis of the filamentous gametophyte (Fig. 1W, X).

Notably and unlike other homosporous-fern gametophytes, young gametophytes of Colysis do not become cordate but are instead ovobate with no apparent notch, i.e. a depressed portion. The ovobate shape may be due to merophytes surrounding the apical cell. In Colysis gametophytes, anticlinal and periclinal divisions occur regularly so each merophyte keeps pace to maintain a rectangular outline (Fig. 1K–N).

Loss of apical cells and further growth by marginal meristems

At around 40 DAS, the apical cell that produced five or six (nine in one extreme case) derivative cells stops dividing at two lateral walls, and instead undergoes periclinal division to create an outer (surface) rectangular and an inner equilateral triangular cell (Fig. 1M, N). By this stage, the apical cell seems to have ceased functioning as the initial cell. Both the outer rectangular and the inner triangular cells continue dividing in the periclinal and anticlinal planes to form a large merophyte maintaining an overall triangular shape (Fig. 2A–D). Other merophytes surrounding the triangular merophyte also expand in area due to an increase in cell number and cell expansion, contributing to gametophyte growth (Fig. 2D). Growing gametophytes appear to have no growth centre after losing their apical cell.

Gametophyte development after loss of the apical-cell-based meristem of Colysis decurrens. Epi-illuminated micrographs (A, C, F, H), micrograph taken under transmitted light (E) and line drawings (B, D, G, I). Numerals at the upper left corners indicate...

After a short period of growth with no apical-cell-based meristem, a new meristematic area is distinguishable in or next to the apical-cell-derived triangular merophyte (Fig. 2C, D). This marginal meristem enlarges to cover the anterior margin of the gametophyte (Fig. 2E, F). It is composed of rectangular cells with outermost (marginal) longest cells (31·0 ± 5·1 µm) and inner shorter cells behind them (25·3 ± 5·3 µm; Figs 2F, H and 3E). Cell lineage analyses of growing gametophytes shows that both the outermost and the inner cells in the meristem undergo cell division in the periclinal and anticlinal planes to form rectangular cell packets (merophytes) with larger merophytes near the margin (Fig. 2G, I). However, even at the margin, no merophytes are substantially larger than others (Fig. 2I), indicating that there is no growth centre with a much higher division rate in the meristem. This behaviour is similar to that in young leaf laminae, and such the structure is described here as a marginal meristem.

The marginal meristem expands further in area through growth, but it does not cover the gametophyte margin entirely. Cells in the proximal periphery of the marginal meristem stop dividing and become fully differentiated and remain as an inactive posterior portion of the thallus (Fig. 2H, I). Therefore, young gametophytes with marginal meristems first become ovobate (Fig. 3A), elongate ovobate (Fig. 2E) and then strap-shaped (Fig. 4A). At the marginal meristem stage, rhizoids are not restricted to the posterior region, but arise from cells at the inner parts of the gametophytes (Fig. 3B, F).

Gametophytes with marginal meristems of Colysis decurrens. Numerals at the upper left corners indicate DAS. (A–C) Micrographs of the same gametophyte undergoing branching by cessation of marginal meristem growth in the centre (arrowhead). (D,...

Mature Colysis decurrens gametophytes with cushion meristems. All gametophytes (A, F, G, J, K, M) are well-grown branched lobes that have been produced by terminal branching of the original gametophyte (not shown). Micrographs taken with transmitted light...

Gametophytes often branch terminally by division of the marginal meristem. Occasionally when the marginal meristem extends in area, meristematic activity stops in the middle of the meristem (Fig. 3B). In these cases, both meristematic areas separated by intervening differentiated cells maintain meristematic activity and grow further to give rise to independent lobes (Fig. 3C). Finally, the gametophyte branches in two. Branched lobes grow equally or unequally depending on the individual.

Mature gametophytes

At around 50 DAS, antheridia begin forming from the relatively basal inner portion of the gametophytes, which are one cell thick and still at the marginal meristem stage (Fig. 3F, G). In better developed gametophytes, antheridia are scattered in the middle along the long axis of the gametophyte. When the gametophyte starts forming archegonia, functional antheridia, when present, are restricted to the posterior portion of the thallus. No antheridia were observed on any branched gametophytes during the course of the study.

The archegonia form first at 55–68 DAS for non-branched gametophytes (observations from seven individuals) and 99–105 DAS for branched gametophytes (two individuals) from a small cushion two layers thick behind the anterior margin (Fig. 4A–E). As the gametophyte grows further, the small cushion becomes extended and narrowly elongated, forming more archegonia in vertical lines (Fig. 4J). Concomitantly, the gametophyte anterior margin shape changes from round (Fig. 4A) to flat (Fig. 4K), and finally becomes slightly depressed, forming a very shallow notch (Fig. 4F, G, J). In the present culture condition, archegonia arose at both upper (dorsal) and lower (ventral) surfaces of the cushion two or three cells thick (Fig. 4I).

Notably, no matter how large the gametophyte becomes, the distance between the anterior margin and cushion starting point, i.e. first paradermal division (parallel to the substratum), remains nearly the same, i.e. about ten cells behind the anterior margin (Fig. 4D, I). This indicates that the cushion is formed by the activity of a meristem that occupies the region anterior to the cushion (Fig. 4H). In this context, this meristem is here called the cushion meristem.

Young cushion meristems are constructed of vertical cell files that are clearly recognizable probably due to recent frequent divisions in the periclinal plane (Fig. 4L). Close-up views of the meristem show that somewhat longer outermost cells divide both in an unequal periclinal plane to produce small cells proximally and slightly longer cells distally, as well as in an anticlinal plane to produce long outermost cells horizontally (inset to Fig. 4L). In mature cushion meristems, the marginal and submarginal cells undergo anticlinal cell divisions more frequently than inner cells, so the number of cell files increases in the horizontal direction near the anterior margin (Fig. 4H). As a result, the mature cushion meristem often becomes triangular in outline (Fig. 4H). The cushion meristem sometimes produces cells rapidly in the horizontal (lateral) and vertical direction, so growth is out of line with the neighbouring area. In this case, the cushion meristem forms either a slightly protruding anterior margin (Fig. 4N) or an enlarged part growing away from the substrate (agar) surface (data not shown).

A gametophyte with a newly initiating cushion meristem has the round anterior margin typical of the marginal meristem stage as mentioned above (Fig. 4A), and there are no gametophytes with both round marginal and cushion meristems near each other. These facts suggest that the cushion meristem may be initiated through changes in the marginal meristem that modify its shape and organization. To clarify the initiation of the cushion meristem, the typical cushion and marginal meristems were first compared morphometrically (for ten and five individuals, respectively) using median longitudinal sections: (1) the thickness of the inner cells is almost identical at 45·2 ± 3·5 vs. 42·6 ± 3·0 µm for the cushion vs. marginal meristems, respectively; (2) the outermost cells are similarly longer than inner cells (compare Fig. 2H with Fig. 4L); (3) the inner cells of the cushion meristem are significantly shorter than those of the marginal meristem (19·0 ± 3·7 vs. 25·3 ± 5·3 µm, respectively, Student's t-test P < 0·05), while outermost cells are not (28·0 ± 3·0 vs. 31·0 ± 5·1 µm); and (4) the length ratio of the outermost to inner cells is larger in the cushion (28·0/19·0 µm = 1·47) than in the marginal meristem (31·0/25·3 µm = 1·23). In summary, the cushion meristem can be distinguished from the marginal meristem by larger length ratios of the outermost to inner cells.

Next, we examined a cushion meristem that was just initiating and was not yet arranged in clear cell files for one individual (Fig. 4A–E). Two serial longitudinal sections of the meristem show that the inner cells (15·5 ± 3·1 µm long, Fig. 4D, and 12·5 ± 5·4 µm long, Fig. 4E) anterior to the cushion are more similar in size to those of the typical cushion meristem (19·0 ± 3·7 µm long, n = 10) than to the marginal meristem (25·3 ± 5·3 µm long, n = 5). In contrast, inner cells in both lateral sides of the initiating cushion meristem are longer (21·8 ± 5·2 µm long, Fig. 4C) and comparable with cells of the marginal meristems (25·3 ± 5·3 µm long, n = 5). The length ratio of the outermost to inner cells is 1·01 (22·0/21·8 µm), 1·08 (16·9/15·7 µm) and 1·53 (21·4/14·0 µm) in Fig. 4C, D and E, respectively. This ratio 1·01 (Fig. 4C) is similar to the length ratio of the outermost to inner cells of the marginal meristem (31·0/25·3 µm = 1·23), and the ratio 1·53 (Fig. 4E) is similar to that of the cushion meristem (28·0/19·0 µm = 1·47), supporting the proposal that the cushion meristem may have been established within the marginal meristem by changes in frequency and orientation of cell divisions.

In Colysis gametophytes, the cushion is usually formed in line but intermittently with intervening one-cell-thick regions (Fig. 4K, M). A gametophyte in Fig. 4K has just begun to initiate the archegonia after a long interruption of cushion formation. Another gametophyte (Fig. 4M) bears two cushion meristems with newly initiated archegonia of their own (Fig. 4N), with the single cushion on the basal common part of the gametophyte. There are no gametophytes with cushion meristems not accompanied by cushions. As shown in Fig. 3A–C, the gametophyte branches only when elongating behind a typical, rounded marginal meristem, and hence the gametophyte in Fig. 4M with a long extension and no cushion is assumed to have gained its marginal meristem after ceasing cushion formation. It seems likely that marginal and cushion meristems appear repeatedly alternately in a growing strap-shaped gametophyte of C. decurrens.

DISCUSSION

Three distinct meristems in Colysis gametophytes

Strap-shaped gametophytes of Colysis, Leptochilus and Paraleptochilus have been described as exhibiting the Kaulina type of development (Nayar, 1962, 1963b; Pal and Pal, 1962; Nayar and Kaur, 1971), in which neither apical cell nor organized meristems (pluricellular meristem) are ever established (Nayar and Kaur, 1969). Contrary to this idea, our cell-lineage analysis of C. decurrens clearly shows that both apical-cell-based meristems and organized meristems are produced in gametophytes.

The apical cell cuts off several derivatives as the single initial cell and then disappears by periclinal division. The number of derivatives formed by the apical cell before its disappearance (5–9 in C. decurrens) is similar to that of other cordiform gametophytes (6–8 in Lygodium japonicum by N. Takahashi, five and six in Osmunda japonica by A. Inoue, unpubl. data). Our preliminary studies show that strap- or ribbon-shaped gametophytes in vittarioids and some genera of Polypodiaceae similarly have single apical cells when young, suggesting that the apical cell is necessary for early growth of fern gametophytes regardless of gametophyte morphology.

In Colysis, an organized multicellular meristem, described here as a cushion meristem, arises later at the reproductive stage. This meristem shows an organization similar to a multicellular meristem (pluricellular meristem, sensuNayar and Kaur, 1971) located in the notch of cordiform gametophytes: a row of several narrow initial cells at the anterior end elongates parallel to the long axis of the gametophyte and actively divides at walls parallel to either lateral or basal walls. Furthermore, the Colysis cushion meristem shares another unique character with the multicellular meristem of cordiform gametophyte: cells proximally cut off from initial cells undergo divisions in a plane parallel to the substratum to form the multi-layered cushion. These facts suggest that the Colysis cushion meristem is similar to the multicellular meristem typical of cordiform gametophytes. In this context, the strap-shaped Colysis gametophytes may have evolved from ancestral cordiform gametophytes by insertion of the marginal meristem phase between the apical-cell-based and the multicellular meristem phases.

However, it might be possible that the Colysis cushion meristem is an altered or modified marginal meristem, because it is developed from the marginal meristem, and when cushion production ceases, the apex regains the rounded aspect of a typical, non-cushion-producing marginal meristem. Wagner and Farrar (1976) also reported that production of an intermittent cushion in non-cordiform gametophytes of Hyalotricha (Polypodiaceae) seems to be correlated with width (robustness) of its gametophyte apex; when growing conditions are poor, the gametophyte apex narrows and ceases production of an archegonial cushion. It could be interpreted that the Colysis marginal meristem has gained the ability to form cushions but it is more parsimonious to suggest that the ability to form the cushion has been retained in Colysis gametophytes during evolution from ancestral cordiform gametophytes.

The presence of both the apical-cell-based meristem and the multicellular meristem may have been overlooked in earlier Colysis research because growing gametophytes have an anterior end that is round, flat or shallowly notched accompanied by much less developed wing portions. The difference in wing shapes and resultant difference in depth of the notch between strap-shaped and cordiform gametophytes is due to the different shapes of merophytes as a whole constructing the wings; Colysis merophytes are rectangular, but those of cordiform gametophytes are fan-shaped with the widest part at the outermost part in cordiform gametophytes (Döpp, 1927 for Onoclea, Cystopteris, Aspidium and others). In addition, at the multicellular meristem stage, cells behind the outermost initial cells divide in the periclinal plane more frequently in Colysis gametophytes than in the cordiform gametophytes of other ferns, resulting in cushion formation further from the anterior end than in cordiform gametophytes: nearly ten cells (C. decurrens in the present study; Stokey and Atkinson, 1958a for Elaphoglossum angustissimum) vs. one or two cells (cordiform gametophytes, e.g. Döpp, 1927 for Onoclea struthiopteris; Stokey, 1945 for Dipteris conjugata).

The marginal meristem is the most important feature distinguishing the strap-shaped gametophyte of C. decurrens from the cordiform gametophytes of other ferns. The occurrence of the so-called marginal meristem has been noted previously for ribbon-shaped gametophytes, but that meristem was an apical meristem that resided in the anterior margin (Farrar et al., 2008). However, the marginal meristem of Colysis is easily distinguished from the apical meristem (the apical-cell-based and the multicellular meristems) in having no growth centre and in not producing the cushion. This meristem is comparable in its organization to the marginal meristem of fern leaves where the marginal meristem consists of conspicuous marginal initials and submarginal cells, and undergoes fractionation to form pinnae (Hagemann, 1965, 1984; Mueller, 1982). Fractionation of the meristem is similarly involved in gametophyte branching in Colysis due to cessation of meristematic activity in the centre of the gametophyte.

There are some developmental data for strap- and ribbon-shaped gametophytes in Hymenophyllaceae (Stokey, 1948), Vittariaceae (Farrar, 1974), grammitids (Stokey and Atkinson, 1958b; Dassler and Farrar, 1997) and other Polypodiaceae (Masuyama, 1975). Some figures presented by these authors suggest the presence of the apical-cell-based, marginal and multicellular meristems, but the data are very fragmentary. Extensive studies of strap- and ribbon-shaped fern gametophytes focusing on meristem behaviour would provide a better understanding of the morphological evolution of such unusually shaped gametophytes.

Evolution and ecology of strap-shaped gametophytes

Nayar (1962) reported that Colysis gametophytes take more than 1 year to form antheridia and more than 2 years to form archegonia (on Knop's agar medium). The present study showed archegonia forming at almost 4 months (using Parker and Thompson's agar medium). The time until archegonia are formed may depend on culture medium and/or conditions, but 4 months is longer than the time taken for archegonia to form in culture on the cordiform gametophytes of other ferns (30 DAS in Lygodium japonicum cultured using the same medium and photoperiod as C. decurrens, N. Takahashi, unpubl. data). Perhaps the longer period of vegetative growth of C. decurrens is partly caused by the insertion of the marginal-meristem phase between the apical-cell-based and pluricellular meristem phases.

A gemmiferous strap gametophyte is better adapted to epiphytic habitats, because the continuous production and dispersal of gemmae may support metapopulation dynamics whereby new habitats can be colonized as older sites become unsuitable, thus accommodating individual population extinction (Farrar et al., 2008). Our studies of C. decurrens to date have revealed no gemmae, although Nayar and Kaur (1971) reported gemmae in all genera of ribbon-like gametophytes of Polypodiaceae, so a longer term study (and observations of gametophytes from natural substrata) would be interesting. However, regardless of whether gemmae occur or not, the indefinite longevity of all perennial gametophytes must be especially significant in colonization of new habitats that are kilometres or thousands of kilometres from the spore source (Rumsey et al., 1998; Dassler and Farrar, 2001). The Colysis gametophyte often reverts to the marginal meristem from the cushion meristem phase. As gametophytes do not form archegonia in the marginal meristem phase, retrieval of the marginal meristem allows the gametophyte to live indefinitely and survive adverse environmental conditions.

In conclusion, strap-shaped gametophytes of C. decurrens are formed by three different developmental phases, each characterized by its own meristem: (1) the apical-cell-based meristem responsible for early growth, (2) the marginal meristem responsible for further growth involving terminal branching of gametophytes and (3) the multicellular cushion meristem for reproductive growth forming the cushion with archegonia. The strap-shaped gametophyte may have evolved from insertion of the marginal meristem phase between the apical-cell-based and multicellular meristem phases, the latter of which is probably common to cordiform gametophytes of other ferns. The marginal meristem repeatedly alternates with the cushion meristem phase, resulting in indefinite gametophyte growth with intermittent cushion formation.

ACKNOWLEDGEMENTS

We thank Dr Atsushi Ebihara of the Department of Botany of the National Museum of Nature and Science, Tokyo, and Professor Hirokazu Tsukaya of Gradate School of Science, University of Tokyo, for suggesting the scientific name of Colysis decurrens, and helping in spore collection and fieldwork, respectively. We also thank Professor Donald R. Farrar and an anonymous referee for detailed and helpful suggestions and comments. This study was supported by Grants-in-Aid for Scientific Research, Japan (grant number 16370046).